Anaplastic lymphoma kinase (ALK) is an orphan receptor tyrosine kinase (RTK) originally identified as part of the nucleophosmin (NPM)-ALK fusion gene in anaplastic large cell lymphoma (ALCL) with a t(2;5) chromosomal translocation (Morris, S. W. et al., Science, 1994, 263, 1281-1284; Shiota, M. et al., Oncogene, 1994, 9, 1567-1574). ALK expression is mainly restricted to the central and peripheral nervous systems, implicating a potential role in the physiological development and function of the nervous system (Iwahara, T. et al., Oncogene, 1997, 14, 439-449; Morris, S. W., et al., Oncogene, 1997, 14, 2175-2188). ALK knockout mice possess a full life span and have no overt abnormalities (Webb, T. R. et al., Expert Rev. Anti-cancer Ther., 2009, 9, 331-356), suggesting ALK inhibition could be well tolerated without severe adverse effects. While the physiological role of ALK receptor has not been well defined, involvement of ALK in the oncogenesis of various human cancers has been well documented and characterized. Besides NPM-ALK, various other ALK fusion genes were subsequently detected in ALCL, inflammatory myofibroblastic tumor (IMT), diffuse large B-cell lymphoma (DLBCL), systemic histiocytosis, and most notably, in non-small cell lung cancer (NSCLC), resulting in the generation of oncogenic ALK fusion proteins with constitutive phosphorylation/activation of ALK, which plays causative role in tumorgenesis by aberrant phosphorylation of intracellular downstream substrates (Webb, T. R. et al., Expert Rev. Anti-cancer Ther., 2009, 9, 331-356; Palmer, R. H. et al., Biochem. J., 2009, 420, 345-361; Chiarle, R. et al., Nature Rev. Cancer, 2008, 8, 11-23; Mano H., Cancer Sci., 2008, 99, 2349-2355). In NSCLC, at least seven isoforms of an oncogenic fusion gene comprised of portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and ALK gene were identified in about 3-15% patients examined (Soda, M. et al., Nature, 2007, 448, 561-566; Choi Y. L. et al., Cancer Res., 2008, 68, 4971-4976; Takeuchi, K. et al., Clin. Cancer Res., 2009, 15, 3143-3149). Experimental data indicate that inhibition of ALK could markedly impair the growth of ALK-positive lymphoma and lung cancer cells in vitro and in vivo, indicating that ALK-positive ALCL and NSCLC cells displayed “ALK oncogene addiction” (Piva, R. et al., Blood, 2006, 107, 689-697; Wan, W. et al., 2006; Galkin, A. V. et al., Proc. Natl. Acad. Sci. USA, 2007, 104, 270-275; Christensen, J. G. et al., Mol. Cancer. Ther., 2007, 6, 3389-3395; Soda, M. et al., Proc. Natl. Acad. Sci. USA, 2008, 105, 19893-19897; Koivunen, J. P. et al., Clin. Cancer Res., 2008, 14, 4275-4283). Recently, it has also been reported that germline mutations in ALK are the cause of most hereditary neuroblastoma cases, and ALK activation by mutation and/or gene amplification is functionally relevant in high-risk sporadic neuroblastoma (Mosse, Y. P. et al., Nature, 2008, 455, 930-936; Chen, Y. et al., Nature, 2008, 455, 971-974; George, R. E. et al., Nature, 2008, 455, 975-978; Janoueix-Lerosey, I. et al., Nature, 2008, 455, 967-970; McDermott, U. et al., Cancer Res., 2008, 68, 3389-3395; Passoni, L. et al., Cancer Res., 2009, 69, 7338-7346). Attenuation and inhibition of ALK activating mutants or wild type (WT) receptor resulted in profound growth inhibition in human neuroblastoma cell lines (Mosse, Y. P. et al., Nature, 2008, 455, 930-936; Chen, Y. et al., Nature, 2008, 455, 971-974; George, R. E. et al., Nature, 2008, 455, 975-978; Janoueix-Lerosey, I. et al., Nature, 2008, 455, 967-970; McDermott, U. et al., Cancer Res., 2008, 68, 3389-3395; Passoni, L. et al., Cancer Res., 2009, 69, 7338-7346), indicating that the ALK receptor, either the activating mutants or overexpressed WT form, is a critical player in neuroblastoma development. Altogether, these findings indicate that ALK is a major therapeutic target for human cancers and inhibition of ALK with a small molecule ALK inhibitor would offer a potentially more effective and less toxic therapy for patients with ALK-positive tumors than conventional chemotherapy.
ALK belongs to the insulin receptor (IR) RTK superfamily, which includes insulin-like growth factor-1 receptor (IGF-1R), insulin related receptor (IRR) and leukocyte tyrosine kinase (LTK) (Iwahara, T. et al., Oncogene, 1997, 14, 439-449; Morris, S W. et al., Oncogene, 1997, 14, 2175-2188). Of these IR family members, inhibition of IR itself poses a potential liability due to its involvement in glucose uptake and metabolism (Espinal, Trends Biochem Sci, 1988, 13, 367-368; Saperstein, R. et al., Biochemistry, 1989, 28, 5694-5701). Development of ALK inhibitors with selectivity over IR is, therefore, desirable.
Focal adhesion kinase (FAK) is an evolutionarily conserved non-receptor tyrosine kinase localized at focal adhesions, sites of cellular contact with the extracellular matrix that functions as a critical transducer of signaling from integrin receptors and multiple receptor tyrosine kinases (Parsons, J. T. et al., Clin. Cancer Res., 2008, 14, 627-632; Han, E. K. and McGonigal, T., Anticancer Agents Med. Chem. 2007, 7: 681-684; Schwock, J. et al., Expert Opin. Ther. Targets, 2010, 14, 77-94). The integrin-activated FAK forms a binary complex with Src which can phosphorylate other substrates and trigger multiple signaling pathways. Given the central role of FAK in mediating signal transduction with multiple SH2- and SH3-domain effector proteins (Mitra, S. K. et al., Mol. Cell Biol., 2005, 6, 56-68), activated FAK plays a central role in mediating cell adhesion, migration, morphogenesis, proliferation and survival in normal and malignant cells (Mitra, S. K. et al., Mol. Cell Biol., 2005, 6, 56-68; McClean, G. W. et al., Nature Rev. Cancer, 2005, 5, 505-515; Han, E. K. and McGonigal, T., Anticancer Agents Med. Chem. 2007, 7: 681-684; Chatzizacharias, N. A. et al., Histol. Histopathol., 2008, 23, 629-650). Compared to normal quiescent cells, FAK over-expression and activation is a hallmark of multiple solid tumors, particularly those with a propensity for bone metastasis, specifically breast cancer, ovarian cancer, NSCLC, prostate cancer, and head/neck squamous cell carcinoma (HNSCC) (Zhao, J. and Guan J. L., Cancer Metastasis Rev., 2009, 28, 35-49.; Schwock, J. et al., Expert Opin. Ther. Targets, 2010, 14, 77-94). Moreover, FAK over-expression and activation mediate anchorage-independent cell survival and are associated with an enhanced invasive and metastatic phenotype and tumor angiogenesis in these malignancies (Owens, L. V. et al., Cancer Res., 1995, 55, 2752-2755., Kornberg, I. J., et al., Head and Neck, 1998, 20: 634-639; McClean, G. W. et al., Nature Reviews Cancer, 2005, 5, 505-515; Han, E. K. and McGonigal, T. Anticancer Agents Med. Chem. 2007, 7: 681-684; Chatzizacharias, N. A. et al., Histol. Histopathol., 2008, 23, 629-650). Elevated FAK levels in tumors are often caused by amplification of the FAK gene locus i.e. in breast carcinomas, and the critical role of FAK in the metastatic progression of breast cancer has been demonstrated pre-clinically in conditional knockout studies (van Nimwegen, M. J. et al., Cancer Res., 2005, 65, 4698-4706.; Pylayeva, Y. et al., J. Clin. Invest., 2009, 119, 252-266). FAK activation also protects tumor cells from chemotherapy-induced apoptosis, contributing further to tumor survival and resistance (Han, E. K. and McGonigal, T. Anticancer Agents Med. Chem. 2007, 7, 681-684; Halder, J. et al., Clin. Cancer Res., 2006, 12, 4916-4924). Multiple proof-of-concept studies conducted in various solid tumors using siRNA (Halder, J. et al., Clin. Cancer Res., 2006, 12, 4916-4924), dominant-negative FAK (Kohno, M. et al, Int. J. Cancer. 2002, 97, 336-343) and small molecule FAK inhibitors (Halder, J. et al., Cancer Res., 2007, 67, 10976-10983; Roberts, W. G. et al., Cancer Res., 2008, 68, 1935-1944.; Bagi, C. M. et al., Cancer, 2008, 112, 2313-2321) have provided pre-clinical support for the therapeutic utility of FAK inhibition as an anti-tumor/anti-angiogenic strategy, particularly for androgen-independent prostate cancers, breast cancers, and HNSCC.
The Janus kinase (JAK)/Signal transducers and activators of transcription (STAT) pathway is the major signaling cascade downstream from cytokine receptors and growth factor receptors including growth hormone, prolactin and leptin (Rane, S. G. et al., Oncogene 2002, 21, 3334-3358; Schindler, C. et al., J. Biol. Chem. 2007, 282, 20059-20066; Baker, S. J. et al., Oncogene, 2007, 15, 6724-6737). The signaling cascade consists of the family of non-receptor tyrosine kinases, JAK and transcription factors, STAT. Activated JAK phosphorylate and activate STAT, allowing formation of homo- and heterodimers that translocate to the nucleus to regulate the transcription of STAT-dependent genes. In addition, STAT can be directly phosphorylated by non-receptor tyrosine kinases like Src or Abl. Under normal physiological conditions ligand-dependent activation of JAK/STAT signaling is transient and tightly regulated (Alexander, W. S., Nature Rev. Immunol., 2002, 2, 1-7; Shuai, K. et al., Nature Rev. Immunol., 2003, 3: 900-910). Constitutive activation of JAK and STAT was detected in a wide spectrum of human cancers, both solid and hematopoietic, and often correlated with a more malignant and metastatic phenotype and refractory tumors (Ferrajoli, A. et al., Curr. Cancer Drug Targets, 2006, 6, 671-9; Yu, H. et al., Nature Rev. Cancer, 2004, 4, 97-105). In most tumors JAK2/STAT activation was mediated by a constitutive expression of cytokines (IL-6, IL-4, GM-CSF) and/or by inactivation of endogenous repressors of the JAK/STAT pathway, including members of the suppressor of cytokine signaling (SOCS) family or phosphatase SHP-1 due to promoter methylation or specific deletions (Yoshikava, H. et al., Nature Genetics, 2001, 28, 29-35; Weber, A., at al., Oncogene, 2005, 24, 6699-708; Melzner, I., et al., Oncogene, 2005, 24, 6699-708; Weniger, M. et al., Oncogene, 2006, 25, 2679-84). In some tumors, activating mutations in JAK1 (Flex, E. et al., J. Exp. Med. 2008, 205, 751-758; Hornakova, T. et al., J. Biol. Chem. 2009, 384, 6773-6781), JAK2, JAK3 or JAK2 chimeric molecules were directly implicated in tumorigenesis. In addition, amplification of the JAK2 locus was found in 35% of Hodgkin lymphoma (HL) and 50% of primary mediastinal B-cell lymphoma (PMBL) cases (Melzner, I, et al., Blood, 2005, 105, 2535-2542). The ectopic expression of JAK1, JAK2 and JAK3, as well as STAT3 and STAT5 resulted in oncogenic transformation in recipient cells, demonstrating that the activated JAK2/STAT pathway was sufficient to mediate oncogenesis in various solid and hematological tumors (Bromberg, J. et al., Cell, 1999, 98, 295-303; Knoops, L. et al., Oncogene, 2008, 27, 1511-9; Scheeren, F. A. et al., Blood. 2008, 111, 4706-4715). In multiple studies, inhibition of JAK2/STAT signaling in various tumor cells, including prostate, breast, colon and lung carcinomas, gliomas as well as leukemias and lymphomas resulted in inhibition of growth, induction of apoptosis and suppression of tumor growth in vivo (Yu, H. et al., Nature Rev. Cancer, 2004, 4, 97-105; Li, H. et al., Cancer Res. 2004, 64, 4774-4782; Iwamaru, A. et al., Oncogene, 2007, 26, 2435-2444; Gao, S., et al., J. Clin. Invest., 2007, 117, 3846-3856; Ding, B. et al., Blood, 2008, 111, 1515-1523,). Pre-clinical studies have demonstrated that constitutively activated JAK2/STAT signaling in tumor cells not only promoted uncontrolled cell proliferation and anti-apoptotic signaling, but also mediated tumor immune evasion and angiogenesis (Kortylewski, R., et al., Nat. Med., 2005, 11, 1314-21; Nefedova, Y. et al., Curr. Cancer Drug Targets, 2007, 7, 71-77). Therefore, inhibitors of JAK/STAT signaling would potentially suppress multiple mechanisms underlying tumor formation and progression.
Human cancers are notoriously heterogeneous, even in those well established “oncogene addicted” tumors, since some cancer cells likely contain additional oncogenic event(s) or redundant active signaling pathways which may render the cancer cells less dependent on the primary oncogene for growth and survival (Hanahan, D. and Weinberg, R. A., Cell, 2000, 100, 57-70). Concomitant inhibition of the secondary oncogenic event(s) in those cancer cells would likely lead to increase the efficacy of treatment with a kinase inhibitor, either by combination therapy or developing a small molecule inhibitor against both the primary and secondary targets. For example, FAK was found to be hyperphosphorylated and activated in a majority of human NSCLC cell lines and contributed to promote cancer cell invasion and metastasis (Rikova, K., Cell, 2007, 131, 1190-1203). An ALK inhibitor with concomitant FAK activity may provide additive or synergistic anti-tumor activity against ALK-positive NSCLC, and potentially additional solid and hematological tumors containing functional ALK and FAK. Similarly, JAK2/STAT5 mediated signaling pathway plays an important role in androgen-independent prostate tumorigenesis (Li, H., et al. Cancer Res., 2004, 64, 4774-4782; Dagvadorj, A., et al., Endocrinology, 2007, 148, 3089-3101) and FAK activity was found to be critical for maintaining the invasive and metastatic phenotype of androgen-independent prostate cancer (Owen, L. V. et al, Cancer Res., 1995, 55, 2752-2755; Tremblay, L., et al., Int. J. Cancer, 1996, 68, 164-171). Therefore, there is a compelling rationale for the utility of a dual JAK2/FAK inhibitor to treat this type of cancer.
On the other hand, although kinase inhibitors have been extremely effective in specific patient populations with tumors containing mutated, oncogenic forms of protein tyrosine kinases (PTK), clinical studies thus far have shown that some patients eventually develop resistance to these drugs, either due to the selection of cancer cells with mutations in the targeted PTK or the induction of compensatory oncogenic signaling pathways (Shah, N. P. and Sawyers, C. L., Oncogene, 2003, 22, 7389-7395; Engelman, J. A. and Settleman, J., Curr. Opin. Genet. Develop., 2008, 18, 1-7; Liu, J. et al., Leukemia, 2008, 22, 791-799; Desai, J. et al., Clin. Cancer Res., 2007, 13, 5398-5405; Engelman, J. A. and Janne, P. A., Clin. Cancer Res., 2008, 14, 2895-2899). In that regard, a kinase inhibitor simultaneously inhibiting two or more critical, non-redundant signaling pathways may prevent or decrease the incident of resistant tumors to develop.
A need exists for ALK, FAK and JAK2 inhibitors for use as pharmaceutical agents.